Paul O. Verhoeven

Comparative Evaluation of Six Commercialized Multiplex PCR Kits for the Diagnosis of Respiratory Infections

The molecular diagnosis of respiratory infection can be performed using different commercial multiplex-based PCR kits whose performances have been previously compared individually to those of conventional techniques. This study compared the practicability and the diagnostic performances of six CE-marked kits available in 2011 on the French market, including 2 detecting viruses and atypical bacteria (from Pathofinder and Seegene companies) and 4 detecting only viruses (from Abbott, Genomica, Qiagen and Seegene companies). The respective sensitivity, specificity, accuracy and agreement of each multiplex technique were calculated by comparison to commercial duplex PCR tests (Argene/bioMérieux) used as gold standard. Eighty-eight respiratory specimens with no pathogen (n = 11), single infections (n = 33) or co-infections (n = 44) were selected to cover 9 viruses or groups of viruses and 3 atypical bacteria. All samples were extracted using the NUCLISENS® easyMAG™ instrument (bioMérieux). The overall sensitivity ranged from 56.25% to 91.67% for viruses and was below 50% with both tests for bacteria. The overall specificity was excellent (>94% for all pathogens). For each tested kit, the overall agreement with the reference test was strong for viruses (kappa test >0.60) and moderate for bacteria. After the extraction step, the hands-on time varied from 50 min to 2h30 and the complete results were available in 2h30 to 9 h. The spectrum of tested agents and the technology used to reveal the PCR products as well as the laboratory organization are determinant for the selection of a kit.

Detection and clinical relevance of Staphylococcus aureus nasal carriage: an update

Staphylococcus aureus nasal carriage is a well-defined risk factor of infection with this bacterium. The increased risk of S. aureus infection in nasal carriers is supported by the fact that the strains isolated from both colonization and infection sites are indistinguishable in most of the cases. Persistent nasal carriage seems to be associated with an increased risk of infection and this status could be defined now in clinical routine by using one or two quantitative nasal samples. There is evidence for supporting the detection of nasal carriage of S. aureus in patients undergoing cardiac surgery and in those undergoing hemodialysis in order to implement decolonization measures. More studies are needed to determine which carriers have the highest risk of infection and why decolonization strategies failed to reduce S. aureus infection in some other groups of patients.

An algorithm based on one or two nasal samples is accurate to identify persistent nasal carriers of Staphylococcus aureus

Persistent Staphylococcus aureus nasal carriers are at high risk of S. aureus infection. The present study delineates a simple strategy aimed at identifying rapidly and accurately this subset of subjects for clinical or epidemiological purposes. Ninety healthy volunteers were each identified as persistent, intermittent or non-nasal carriers of S. aureus by using seven specimens sampled over a 5-week period. By reference to this so-called reference standard, six other strategies aimed at simplifying and speeding the identification of persistent carriers and based on the qualitative or quantitative detection of S. aureus in one to three nasal samples were evaluated by the measure of the area under the curve of receiver operating characteristic diagrams. Among strategies using qualitative results, there was no statistical difference between protocols using seven and three samples. A threshold of 10(3) CFU of S. aureus per swab was found capable of defining persistent nasal carriage with a sensitivity of 83.1% and a specificity of 95.6%. These figures reached 95.5% and 94.9%, respectively, by using an algorithm including one or two nasal specimens according to the threshold of 10(3) CFU of S. aureus in the first swab. The latter two strategies were shown to be costly equivalents. The proposed algorithm-based strategy proved to be relevant to identify properly and consistently persistent nasal carriers of S. aureus. However, as it was built from data of healthy volunteers, it needs to be confirmed prospectively on patients potentially at risk for S. aureus infection.

Better Detection of Staphylococcus aureus Nasal Carriage by Use of Nylon Flocked Swabs

ABSTRACT Flocked swabs (Copan) were compared to rayon swabs (Copan) for the nasal detection of Staphylococcus aureus in 90 healthy volunteers sampled sequentially during a 5-week period. The use of flocked swabs improved the number of nasal carriers (P = 0.026), the number of positive specimens (P = 0.01), and the quantity of bacteria in positive samples (P = 0.004).

Nosocomial transmission of NDM-1-producing Escherichia coli within a non-endemic area in France.

Two patients with no travel history and sharing the same room were colonized by the same strain of New Delhi metallo-β-lactamase 1 (NDM-1)-producing Escherichia coli within a geographical area not endemic for this highly multidrug-resistant bacterium. It was documented an absence of an epidemiological and bacteriological link with a third patient returning from India after surgery and found to be infected by an NDM-1-producing Citrobacter strain during the same period. Despite extensive investigation, the source of contamination of the two former patients was not elucidated. This case report illustrates the need of investigating rapidly the emergence of highly multidrug-resistant Enterobacteriaceae, to stop their dissemination in a nosocomial setting.

Epidemiology and microbiological investigations of community-acquired pneumonia in children admitted at the emergency department of a university hospital.

Abstract Background The management of children with community-acquired pneumonia (CAP) is largely influenced by the development of new molecular diagnostic tests that allow the simultaneous detection of a wide range of pathogens. Objectives Evaluation of a diagnostic approach including multiplex PCR assays for revisiting the epidemiology and etiology of CAP in children at hospital. Study design Children of all ages consulting at the Emergency Department of the University hospital of Saint-Etienne, France, during the 2012–2013 winter period were included. In addition to bacterial cultures, the following pathogens were detected using biplex commercially-available rt-PCR tests: adenovirus, respiratory syncytial virus, human metapneumovirus, bocavirus, rhinovirus/enterovirus, coronavirus, influenza viruses A and B, parainfluenza viruses, Mycoplasma pneumoniae and Chlamydophila pneumonia. Results From 85 patients with CAP, at least one pathogen was identified in 81 cases (95.3%), including 4 bacterial exclusive infections (4.7%), 53 viral exclusive infections (62.4%) and 24 mixed infections (28.2%). Coinfection by at least two viruses was observed in 37 cases (43.5%). Mean age was higher in the case of documented bacterial infection (P <0.05). In the subgroup of viral exclusive infection, the mean age of severe cases was 2.0 years vs 3.8 years in mild and moderate cases (P <0.05). Conclusions These findings highlight the huge proportion of CAP of viral origin, the high number of co-infection by multiple viruses and the low number of bacterial CAP, notably in children under 5 years, and address the need to re-evaluate the indications of empiric antimicrobial treatment in this age group.

Staphylococcal vaccine development: review of past failures and plea for a future evaluation of vaccine efficacy not only on staphylococcal infections but also on mucosal carriage

Staphylococcal disease represents a universal burden including acute, life-threatening infections as well as chronic infections usually associated with foreign materials. Infections occur notably in permanent carriers of Staphylococcus aureus. To date, all the attempts to develop an efficacious vaccine against S. aureus have failed. Failures in vaccine clinical trials might be related to a focus on single targets and development of humoral-based vaccines rather than vaccines with a combination of antigens stimulating both humoral and cellular immunity. The end points of these unsuccessful trials were a reduction in mortality or bacteremia, whereas the patient’s decolonization was not assessed. Adopting the latter point of view, the aim of this article is to discuss nasal mucosal decolonization as a complementary marker of vaccine efficacy for clinical research in vaccine development.

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

ABSTRACT A multiplex real-time PCR (quantitative PCR [qPCR]) assay detecting herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA together with an internal control was developed on the BD Max platform combining automated DNA extraction and an open amplification procedure. Its performance was compared to those of PCR assays routinely used in the laboratory, namely, a laboratory-developed test for HSV DNA on the LightCycler instrument and a test using a commercial master mix for VZV DNA on the ABI7500fast system. Using a pool of negative cerebrospinal fluid (CSF) samples spiked with either calibrated controls for HSV-1 and VZV or dilutions of a clinical strain that was previously quantified for HSV-2, the empirical limit of detection of the BD Max assay was 195.65, 91.80, and 414.07 copies/ml for HSV-1, HSV-2, and VZV, respectively. All the samples from HSV and VZV DNA quality control panels (Quality Control for Molecular Diagnostics [QCMD], 2013, Glasgow, United Kingdom) were correctly identified by the BD Max assay. From 180 clinical specimens of various origins, 2 CSF samples were found invalid by the BD Max assay due to the absence of detection of the internal control; a concordance of 100% was observed between the BD Max assay and the corresponding routine tests. The BD Max assay detected the PCR signal 3 to 4 cycles earlier than did the routine methods. With results available within 2 h on a wide range of specimens, this sensitive and fully automated PCR assay exhibited the qualities required for detecting simultaneously HSV and VZV DNA on a routine basis.

Quantification by real-time PCR assay of Staphylococcus aureus load: a useful tool for rapidly identifying persistent nasal carriers

ABSTRACT The Cepheid Xpert MRSA/SA nasal PCR assay was compared to culture for quantifying Staphylococcus aureus load from 104 nasal samples (r = 0.91, P < 0.0001). Using a bacterial load-based algorithm, the test was found able to predict the carrier state in 32 of 35 healthy volunteers (22 persistent and 13 nonpersistent carriers).

Detection of Carbapenemase-Producing Bacteria by Using an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method

ABSTRACT The emergence of carbapenemase-producing bacteria poses a new challenge in the management of antibiotic therapies for patients. This report describes a new method using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for rapid detection of carbapenemase activity in enterobacteria, Pseudomonas aeruginosa, and Acinetobacter baumannii. In a panel of 78 isolates, including 41 carbapenemase-producing strains, the ULPC-MS/MS assay showed 100% agreement with molecular characterization, whereas six carbapenemase-producing isolates were not detected by the modified Hodge test.